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Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma.

ABSTRACT: BACKGROUND:Although mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs. METHODS:In-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data. RESULTS:The approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations. CONCLUSIONS:This study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.

SUBMITTER: Loffler MW

PROVIDER: S-EPMC6492406 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:BackgroundNeoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor, where the neoantigen repertoire has not been investigated. Our aim was to analyze whether tumors in HCC patients contain immunogenic neoantigens suitable for future use in therapeutic vaccination.MethodsWhole-exome sequencing and RNAseq were performed in a cohort of fourteen HCC patients submitted to surgery or liver transplant. To identify mutations, single-nucleotide variants (SNV) originating non-synonymous changes that were confirmed at the RNA level were analyzed. Immunogenicity of putative neoAgs predicted by HLA binding algorithms was confirmed by using in vitro HLA binding assays and T-cell stimulation experiments, the latter in vivo, by immunizing HLA-A*02.01/HLA-DRB1*01 (HHD-DR1) transgenic mice, and in in vitro, using human lymphocytes.ResultsSequencing led to the identification of a median of 1217 missense somatic SNV per patient, narrowed to 30 when filtering by using RNAseq data. A median of 13 and 5 peptides per patient were predicted as potential binders to HLA class I and class II molecules, respectively. Considering only HLA-A*02.01- and HLA-DRB1*01-predicted binders, 70% demonstrated HLA-binding capacity and about 50% were immunogenic when tested in HHD-DR1 mice. These peptides induced polyfunctional T cells that specifically recognized the mutated but not the wild-type sequence as well as neoantigen-expressing cells. Moreover, coimmunization experiments combining CD8 and CD4 neoantigen epitopes resulted in stronger CD8 T cell responses. Finally, responses against neoantigens were also induced in vitro using human cells.ConclusionThese results show that mutations in HCC tumors may generate immunogenic neoantigens with potential applicability for future combinatorial therapeutic strategies.

Project description:BackgroundHepatocellular carcinoma (HCC) was the sixth common malignancies characteristic with highly aggressive in the world. It was well established that tumor mutation burden (TMB) act as indicator of immunotherapeutic responsiveness in various tumors. However, the role of TMB in tumor immune microenvironment (TIME) is still obscure.MethodThe mutation data was analyzed by employing "maftools" package. Weighted gene co-expression network analysis (WGCNA) was implemented to determine candidate module and significant genes correlated with TMB value. Differential analysis was performed between different level of TMB subgroups employing R package "limma". Gene ontology (GO) enrichment analysis was implemented with "clusterProfiler", "enrichplot" and "ggplot2" packages. Then risk score signature was developed by systematical bioinformatics analyses. K-M survival curves and receiver operating characteristic (ROC) plot were further analyzed for prognostic validity. To depict comprehensive context of TIME, XCELL, TIMER, QUANTISEQ, MCPcounter, EPIC, CIBERSORT, and CIBERSORT-ABS algorithm were employed. Additionally, the potential role of risk score on immune checkpoint blockade (ICB) immunotherapy was further explored. The quantitative real-time polymerase chain reaction was performed to detect expression of HTRA3.ResultsTMB value was positively correlated with older age, male gender and early T status. A total of 75 intersection genes between TMB-related genes and differentially expressed genes (DEGs) were screened and enriched in extracellular matrix-relevant pathways. Risk score based on three hub genes significantly affected overall survival (OS) time, infiltration of immune cells, and ICB-related hub targets. The prognostic performance of risks score was validated in the external testing group. Risk-clinical nomogram was constructed for clinical application. HTRA3 was demonstrated to be a prognostic factor in HCC in further exploration. Finally, mutation of TP53 was correlated with risk score and do not interfere with risk score-based prognostic prediction.ConclusionCollectively, a comprehensive analysis of TMB might provide novel insights into mutation-driven mechanism of tumorigenesis further contribute to tailored immunotherapy and prognosis prediction of HCC.

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Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma.

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